Biomarkers of Efficacy and Safety of the Academic BCMA-CART ARI0002h for the Treatment of Refractory Multiple Myeloma.

PURPOSE: B-cell maturation antigen (BCMA)-chimeric antigen receptor T-cells (CART) improve results obtained with conventional therapy in the treatment of relapsed/refractory multiple myeloma. However, the high demand and expensive costs associated with CART therapy might prove unsustainable for health systems. Academic CARTs could potentially overcome these issues. Moreover, response biomarkers and resistance mechanisms need to be identified and addressed to improve efficacy and patient selection. Here, we present clinical and ancillary results of the 60 patients treated with the academic BCMA-CART, ARI0002h, in the CARTBCMA-HCB-01 trial. PATIENTS AND METHODS: We collected apheresis, final product, peripheral blood and bone marrow samples before and after infusion. We assessed BCMA, T-cell subsets, CART kinetics and antibodies, B-cell aplasia, cytokines, and measurable residual disease by next-generation flow cytometry, and correlated these to clinical outcomes. RESULTS: At cut-off date March 17, 2023, with a median follow-up of 23.1 months (95% CI, 9.2-37.1), overall response rate in the first 3 months was 95% [95% confidence interval (CI), 89.5-100]; cytokine release syndrome (CRS) was observed in 90% of patients (5% grades ≥3) and grade 1 immune effector cell-associated neurotoxicity syndrome was reported in 2 patients (3%). Median progression-free survival was 15.8 months (95% CI, 11.5-22.4). Surface BCMA was not predictive of response or survival, but soluble BCMA correlated with worse clinical outcomes and CRS severity. Activation marker HLA-DR in the apheresis was associated with longer progression-free survival and increased exhaustion markers correlated with poorer outcomes. ARI0002h kinetics and loss of B-cell aplasia were not predictive of relapse. CONCLUSIONS: Despite deep and sustained responses achieved with ARI0002h, we identified several biomarkers that correlate with poor outcomes.

Engineered T cells secreting anti-BCMA T cell engagers control multiple myeloma and promote immune memory in vivo.

Multiple myeloma is the second most common hematological malignancy in adults and remains an incurable disease. B cell maturation antigen (BCMA)-directed immunotherapy, including T cells bearing chimeric antigen receptors (CARs) and systemically injected bispecific T cell engagers (TCEs), has shown remarkable clinical activity, and several products have received market approval. However, despite promising results, most patients eventually become refractory and relapse, highlighting the need for alternative strategies. Engineered T cells secreting TCE antibodies (STAb) represent a promising strategy that combines the advantages of adoptive cell therapies and bispecific antibodies. Here, we undertook a comprehensive preclinical study comparing the therapeutic potential of T cells either expressing second-generation anti-BCMA CARs (CAR-T) or secreting BCMAxCD3 TCEs (STAb-T) in a T cell-limiting experimental setting mimicking the conditions found in patients with relapsed/refractory multiple myeloma. STAb-T cells recruited T cell activity at extremely low effector-to-target ratios and were resistant to inhibition mediated by soluble BCMA released from the cell surface, resulting in enhanced cytotoxic responses and prevention of immune escape of multiple myeloma cells in vitro. These advantages led to robust expansion and persistence of STAb-T cells in vivo, generating long-lived memory BCMA-specific responses that could control multiple myeloma progression in xenograft models, outperforming traditional CAR-T cells. These promising preclinical results encourage clinical testing of the BCMA-STAb-T cell approach in relapsed/refractory multiple myeloma.

Advancing CART therapy for acute myeloid leukemia: recent breakthroughs and strategies for future development.

Chimeric antigen receptor (CAR) T therapies are being developed for acute myeloid leukemia (AML) on the basis of the results obtained for other haematological malignancies and the need of new treatments for relapsed and refractory AML. The biggest challenge of CART therapy for AML is to identify a specific target antigen, since antigens expressed in AML cells are usually shared with healthy haematopoietic stem cells (HSC). The concomitant expression of the target antigen on both tumour and HSC may lead to on-target/off-tumour toxicity. In this review, we guide researchers to design, develop, and translate to the clinic CART therapies for the treatment of AML. Specifically, we describe what issues have to be considered to design these therapies; what in vitro and in vivo assays can be used to prove their efficacy and safety; and what expertise and facilities are needed to treat and manage patients at the hospital.

First report of CART treatment in AL amyloidosis and relapsed/refractory multiple myeloma.

Multiple myeloma (MM) remains incurable despite the number of novel therapies that have become available in recent years. Occasionally, a patient with MM will develop an amyloid light-chain (AL) amyloidosis with organ dysfunction. Chimeric antigen receptor T-cell (CART) therapy has become a promising approach in treating hematological malignancies. Our institution has developed a second-generation B-cell maturation antigen (BCMA)-CART which is currently being tested in a clinical trial for relapsed/refractory MM.We present the first reported case, to our knowledge, of a patient with AL amyloidosis and renal involvement in the course of an MM, successfully treated with CART therapy targeting BCMA. The patient received a fractioned dose of 3×106/kg BCMA-CARTs after lymphodepletion. At 3 months from infusion, the patient had already obtained a deep hematological response with negative measurable residual disease by flow cytometry in the bone marrow. After 12 months, the patient remains in hematological stringent complete remission and has achieved an organ renal response with a decrease of 70% of proteinuria.This case suggests that concomitant AL amyloidosis in the setting of MM can benefit from CART therapy, even in patients in which predominant symptoms at the time of treating are caused by AL amyloidosis.

NK cells enhance CAR-T cell antitumor efficacy by enhancing immune/tumor cells cluster formation and improving CAR-T cell fitness.

BACKGROUND: Chimeric antigen receptor (CAR)-T cell immunotherapy has modified the concept of treatment in hematological malignancies. In comparison with pediatric patients, where responses are maintained over many years, older patients, such as those with non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), present lower persistence of CAR-T cells that might be due to decreased fitness of T cells acquired with aging. Moreover, cord blood derived-NK cells (CB-NKs) and CAR-NK cells derived from CB-NK can be used 'off-the-shelf' as immune cells with antitumor properties for the treatment of cancer patients. However, to date, clinical studies have only demonstrated the safety of these therapies but not optimal efficacy. To confront the shortcomings of each therapy, we devised a novel approach consisting of simultaneous (CAR-)NK cell and CAR-T cell administration. In this setting, NK cells demonstrate an important immunoregulation of T cells that could be exploited to enhance the efficacy of CAR-T cells. METHODS: A combinatorial treatment based on either CAR-T and CAR-NK cells or CB-NK and CAR-T cells in two models of NHL and MM was performed. Antitumor efficacy was analyzed in vitro and in vivo, and parameters related to early activation, exhaustion and senescence of T cells were analyzed. RESULTS: We show that CAR-NK cells derived from CB-NK are only effective at high doses (high E:T ratio) and that their activity rapidly decreases over time in comparison with CAR-T cells. In comparison and to exploit the potential of 'off-the-shelf' CB-NK, we demonstrate that a low number of CB-NK in the CAR-T cell product promotes an early activation of CAR-T cells and their migration to MM cells leading to enhanced anti-MM efficacy. Moreover, cytokines related to CRS development were not increased, and importantly, CB-NK enhanced the fitness of both CARpos and CARneg T cells, promoting lower levels of exhaustion and senescence. CONCLUSION: This study demonstrates a relevant immunoregulatory role of CB-NK collaborating with CAR-T cells to enhance their antitumor activity. A novel and different approach to consider in CAR-T cell immunotherapy studies is presented here with the goal to enhance the efficacy of the treatment.

Preclinical development of a humanized chimeric antigen receptor against B cell maturation antigen for multiple myeloma.

Multiple myeloma is a prevalent and incurable disease, despite the development of new and effective drugs. The recent development of chimeric antigen receptor (CAR)-T cell therapy has shown impressive results in the treatment of patients with relapsed or refractory hematological B cell malignancies. In the recent years, B-cell maturation antigen (BCMA) has appeared as a promising antigen to target using a variety of immuno-therapy treatments including CART cells, for MM patients. To this end, we generated clinical-grade murine CART cells directed against BCMA, named ARI2m cells. Having demonstrated its efficacy, and in an attempt to avoid the immune rejection of CART cells by the patient, the single chain variable fragment was humanized, creating ARI2h cells. ARI2h cells demonstrated comparable in vitro and in vivo efficacy to ARI2m cells, and superiority in cases of high tumor burden disease. In terms of inflammatory response, ARI2h cells showed a lower TNFα production and lower in vivo toxicity profile. Large-scale expansion of both ARI2m and ARI2h cells was efficiently conducted following Good Manufacturing Practice guidelines, obtaining the target CART cell dose required for treatment of multiple myeloma patients. Moreover, we demonstrate that soluble BCMA and BCMA released in vesicles impacts on CAR-BCMA activity. In summary, this study sets the bases for the implementation of a clinical trial (EudraCT code: 2019-001472-11) to study the efficacy of ARI2h cell treatment for multiple myeloma patients.

Natural Killer Cells in Immunotherapy: Are We Nearly There?

Natural killer (NK) cells are potent anti-tumor and anti-microbial cells of our innate immune system. They are equipped with a vast array of receptors that recognize tumor cells and other pathogens. The innate immune activity of NK cells develops faster than the adaptive one performed by T cells, and studies suggest an important immunoregulatory role for each population against the other. The association, observed in acute myeloid leukemia patients receiving haploidentical killer-immunoglobulin-like-receptor-mismatched NK cells, with induction of complete remission was the determinant to begin an increasing number of clinical studies administering NK cells for the treatment of cancer patients. Unfortunately, even though transfused NK cells demonstrated safety, their observed efficacy was poor. In recent years, novel studies have emerged, combining NK cells with other immunotherapeutic agents, such as monoclonal antibodies, which might improve clinical efficacy. Moreover, genetically-modified NK cells aimed at arming NK cells with better efficacy and persistence have appeared as another option. Here, we review novel pre-clinical and clinical studies published in the last five years administering NK cells as a monotherapy and combined with other agents, and we also review chimeric antigen receptor-modified NK cells for the treatment of cancer patients. We then describe studies regarding the role of NK cells as anti-microbial effectors, as lessons that we could learn and apply in immunotherapy applications of NK cells; these studies highlight an important immunoregulatory role performed between T cells and NK cells that should be considered when designing immunotherapeutic strategies. Lastly, we highlight novel strategies that could be combined with NK cell immunotherapy to improve their targeting, activity, and persistence.

Development of a Novel Anti-CD19 Chimeric Antigen Receptor: A Paradigm for an Affordable CAR T Cell Production at Academic Institutions.

Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells in vitro, inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation. In vivo, A3B1 CAR T cells are able to fully control disease progression in an NOD.Cg-Prkdc scid Il2rd tm1Wjl /SzJ (NSG) xenograph B-ALL mouse model. Based on the pre-clinical data, we conclude that our CART19 is clearly functional against CD19+ cells, to a level similar to other CAR19s currently being used in the clinic. Concurrently, we describe the implementation of our CAR T cell production system, using lentiviral vector and CliniMACS Prodigy, within a medium-sized academic institution. The results of the validation phase show our system is robust and reproducible, while maintaining a low cost that is affordable for academic institutions. Our model can serve as a paradigm for similar institutions, and it may help to make CAR T cell treatment available to all patients.

CAR-T Cell Therapy: A Door Is Open to Find Innumerable Possibilities of Treatments for Cancer Patients

Seven years ago a chronic lymphocytic leukemia patient was for the first time successfully treated with chimeric antigen receptor (CAR)-modified T cells (CAR-T cells) to target CD19 overexpression in tumor cells. This was the beginning of the development of a new type of immunotherapy treatment in cancer patients. Since then, identification of novel antigens expressed in tumor cells and optimization of both CAR constructs and protocols of administration have opened up new avenues for the successful treatment of other hematological malignancies. However, research still continues to avoid some problems such as toxicities associated with the treatment and to find strategies to avoid tumor cell immune evasion mechanisms. On the other hand, for solid tumors, CAR-T therapy results are still in an early phase. In contrast to hematological malignancies, the complex tumor heterogeneity of solid tumors has led to the research of novel and challenging strategies to improve CAR-T cell activity. Here, we will review the main clinical results obtained with CAR-T cells in hematological malignancies, specifically focusing on CAR-T-19 and CAR-T against B-cell maturation antigen (CAR-T-BCMA). Moreover, we will mention the main problems that decrease CAR-T cell activity in solid tumors and the strategies to overcome them. Finally, we will present some of the first clinical results obtained for solid tumors.

Loss of the Immune Checkpoint CD85j/LILRB1 on Malignant Plasma Cells Contributes to Immune Escape in Multiple Myeloma.

Mechanisms of immune regulation may control proliferation of aberrant plasma cells (PCs) in patients with monoclonal gammopathy of undetermined significance (MGUS) preventing progression to active multiple myeloma (MM). We hypothesized that CD85j (LILRB1), an inhibitory immune checkpoint for B cell function, may play a role in MM pathogenesis. In this study, we report that patients with active MM had significantly lower levels of CD85j and its ligand S100A9. Decreased CD85j expression could also be detected in the premalignant condition MGUS, suggesting that loss of CD85j may be an early event promoting tumor immune escape. To gain insight into the molecular mechanisms underlying CD85j functions, we next enforced expression of CD85j in human myeloma cell lines by lentiviral transduction. Interestingly, gene expression profiling of CD85j-overexpressing cells revealed a set of downregulated genes with crucial functions in MM pathogenesis. Furthermore, in vitro functional assays demonstrated that CD85j overexpression increased susceptibility to T cell- and NK-mediated killing. Consistently, ligation of CD85j decreased the number of PCs from individuals with MGUS but not from patients with MM. In conclusion, downregulation of inhibitory immune checkpoints on malignant PCs may provide a novel mechanism of immune escape associated with myeloma pathogenesis.

Natural Killer Cells: Angels and Devils for Immunotherapy.

In recent years, the relevance of the immune system to fight cancer has led to the development of immunotherapy, including the adoptive cell transfer of immune cells, such as natural killer (NK) cells and chimeric antigen receptors (CAR)-modified T cells. The discovery of donor NK cells' anti-tumor activity in acute myeloid leukemia patients receiving allogeneic stem cell transplantation (allo-SCT) was the trigger to conduct many clinical trials infusing NK cells. Surprisingly, many of these studies did not obtain optimal results, suggesting that many different NK cell parameters combined with the best clinical protocol need to be optimized. Various parameters including the high array of activating receptors that NK cells have, the source of NK cells selected to treat patients, different cytotoxic mechanisms that NK cells activate depending on the target cell and tumor cell survival mechanisms need to be considered before choosing the best immunotherapeutic strategy using NK cells. In this review, we will discuss these parameters to help improve current strategies using NK cells in cancer therapy. Moreover, the chimeric antigen receptor (CAR) modification, which has revolutionized the concept of immunotherapy, will be discussed in the context of NK cells. Lastly, the dark side of NK cells and their involvement in inflammation will also be discussed.